The present invention relates to isolation of messenger RNA (mRNA) from cells and tissues, preparation of cDNA, mRNA libraries and cDNA libraries. All available cDNA libraries to date have been constructed from polyadenylated RNA, on the premise that the majority of mRNA sequences are polyadenylated. Polyadenylated mRNA has typically been isolated by chromatography on oligo(dT). However, two problems have had to be faced by workers seeking to clone and sequence cDNA. The first stems from the fact that polyadenylation occurs at the 3′-ends of RNA and that the 5′ terminal sequences are frequently absent from mRNA and cDNA libraries and are often difficult to obtain even by supplementary means. The second, less obvious problem, is that a significant fraction of mRNAs in a cell at any given time might include mRNAs that are not polyadenylated. The possibility that many mRNAs were simply missed by oligo(dT) isolation has now been confirmed by the results presented herein. The present invention provides methodology for solving both problems, by isolating mRNA based on a common feature of the 5′ end, the m7G cap.
Studies of the process of protein synthesis in eukaryotic cells have shown that initiation of translation (the process of protein synthesis based on sequence information of the mRNA) requires molecular modification of the 5′ end of mRNA. The modifications include the covalent addition of a “cap” of 7-methylguanosine diphosphate (m7GDP) to the 5′ end of mRNA, and the subsequent non-covalent binding of a complex of initiation factors. Watson, J. D. et al. Molecular Biology of the Gene, 4th ed. p. 569 Benjamin, Menlo Park, 1987. The primary component involved in the binding of initiation factors to the capped mRNA is the protein designated eIF4E (initiation factor 4E), which binds directly to the m7GDP of the mRNA cap and then functions to facilitate the binding of other protein initiation factors.
The eIF4E protein has been cloned, sequenced, expressed and purified. Its binding to the cap structure has been studied in detail. Variant structures (mutants) having single amino acid substitutions, have been synthesized; having either enhanced or reduced binding affinity for the m7G cap structure (U.S. Pat. No. 6,232,442). It is clear from a biological perspective that the binding affinity of eIF4E for capped mRNA is a significant factor regulating the rate of protein synthesis in cells. The present invention is a practical application of eIF4E variants having enhanced binding affinity for capped mRNA.
Prior attempts to employ eIF4E as a binding agent to isolate capped mRNA have been reported [Edery (1995) Mol. Cell. Biol. 15:3363-3371]. However, the yield was low, probably because high-affinity eIF4E was not known at the time the work was reported. As a result the binding was less efficient, as comparative studies described herein have shown, and column chromatography was required to effect purification. No comparison with the oligo(dT) method was reported and no follow-up studies have been reported.
The sequence of DNA encoding human eIF4E has been determined [Reychlik, W. et al. (1987) Proc. Natl. Acad. USA 84:945-949]. Yeast eIF4E and a fusion protein of mouse eIF4E have been expressed in E. coli [Edery, I., et al. (1998) Gene 74:517-525; Edery, I., et al. (1995) Mol. Cell. Biol. 15:3363-3371]. Haas, D. W. et al. (1991) Arch. Biochem. Biophys. 284:84-89 reported purification of native eIF4E from erythrocytes. Stern, B. D. et al. (1993) reported isolation of recombinant eIF4E using denaturing concentrations of urea.
The co-crystal structure of eIF4E with m7GDP suggests that eIF4E binds to the 5′ cap mRNA with a π—π stacking interaction between two tryptopan residues, sandwiching the m7G base as well as hydrogen bonds between base and acidic protein side chains. Using site-directed mutagenesis on eIF4E, a π—π stacking interaction between two tryptopan residues (Trp-56/Trp-102) and m7GTP was demonstrated. Additionally, Glu-103 in eIF4E is required for hydrogen bonding to m7G. The m7GTP binding site in mammalian eIF4E resides along the S1-S2 and S3-S4 loops. Previous photolabeling studies of eIF4E with [γ-32P]8-N3GTP demonstrated crosslinking at Lys-119 in the S4-H2 loop distant from the m7GTP binding site. A molecular model based on the cocrystal structure of eIF4E/m7GTP suggested that 8-N3GTP binds to a site occupied by the second nucleotide of mRNA.